The mechanism and evolution of recombinational repair.

Abstract

Recently, hydrogen peroxide (H₂O₂), and its free-radical product the hydroxyl radical (OH·), have been identified as major sources of DNA damage in living organisms. We examined DNA repair of hydrogen peroxide damage, using a standard bacteriophage T4 test system in which several different types of repair could be determined. Post-replication recombinational repair and denV-dependent excision repair had little or no effect on H₂O₂ damage. Also, an enzyme important in repair of H₂O₂-induced DNA damage in the E. coli host cells, exonuclease III, was not utilized in repair of lethal H₂O₂ damage to the phage. However, multiplicity reactivation, a form of recombinational repair between multiply infecting phage genomes, was found to repair H₂O₂ damages efficiently. The RAD52 gene of Saccharomyces cerevisiae and genes 46 and 47 of bacteriophage T4 are essential for most recombination and recombinational repair in their respective organisms. The RAD52 gene was introduced into expression vectors which were used to transform E. coli. RAD52 expression was induced, and its ability to complement either gene 46 or gene 47 phage mutants was determined with respect to phage growth, recombination, and recombinational repair. RAD52 gene expression allowed growth of gene 46 and gene 47 mutants under otherwise restrictive conditions, as measured by plaque formation and burst size. The RAD52 gene also restored the ability of gene 46 and gene 47 mutants to undergo recombination of rII markers. Furthermore, the RAD52 gene restored recombinational repair after UV irradiation of gene 46 and gene 47 mutants. The published DNA sequence of RAD52 was compared with the published sequences of genes 46 and 47. Although overall homologies were only marginally significant, RAD52 and gene 46 had substantial sequence similarity over a limited region. These results indicate that the recombinational repair pathway found in phage T4 may be ubiquitous for DNA damage caused by endogenous exidative reactions. Furthermore, they indicated that an essential element of the recombination mechanism in both procaryotic viruses and eucaryotes arose from a common ancestor. Procaryotes and eucaryotes are thought to have diverged at least one billion years ago. Thus, recombination apparently arose early in evolution.